1. Field of the Invention
The present invention relates to a method of employing enriched speciated isotope spikes in the same speciated form as a specie to be measured regardless of incomplete extraction or the presence of conversion, partial destruction, and instability.
2. Description of the Prior Art
The need for making quantitative determinations of a specie of interest occurs in many contexts including environmental, biological, pharmaceutical and industrial samples and in standard reference materials. For example, certain forms of an element or molecular species may exhibit different toxicities or chemical behaviors from others. Existing techniques, with the exception of electrochemical methods, rely predominately on physical separation and time. They are incapable of determining whether the species cross-over (transformation of one specie form into another), are lost, created or are altered or are completely recovered. Such techniques cannot be used to determine transformation of one species into another, destruction or generation during storage, manipulation and sample preparation during the measurement process, or in the separations that are incomplete or variable in measurement processes.
An example of the criticality of such measurements would be to consider chromium. While Cr(III) is a trace element essential for human health, Cr(VI) is poisonous to humans and most other animals and is also a carcinogen. As a result, the difference between these two species, which resides in the oxidation state of the element, may be of critical importance. While chromatography can be used to separate Cr(III) in time resolution from Cr(VI), as each specie can react with its surroundings and even with separating agencies, the chromatographic separation is only a snapshot in time recording the state of affairs at the end of the manipulation. Each specie may have reacted with many other reagents and transformed during the analysis. There is, therefore, with time resolution, no way of determining how much chromium was actually in each specie when the experiment began or when the sample was actually taken.
Specific species are often required for a particular process. For example, barium is toxic in some compound forms, but is also prescribed for medical diagnostic x-ray tests, usually as barium sulfate in liquid slurry form. The conformation and evaluation of a body""s processing of barium into another specie can be accomplished with isotopically labeled barium sulfate. These studies have been done, but the use of speciated isotope dilution measurements has not been used for such analysis.
Some isotopic tracking has been done for lead due to terrestrially unique and naturally occurring isotopic composition differences of this element. The isotopic ratios can be matched with a particular source to determine the origin of the lead. These measurements are not speciated measurements, but depend on the isotopic ratio differences of the natural material to be detected. This technique has also been used for lead pottery glazes to determine the origin of art objects, however in this case also, naturally occurring isotopic ratios were determined. Lead is a uniquely feasible non-radioactive element to be evaluated as to origin by the isotopic ratio method, as its isotopic ratios change with the amount of uranium mixed with the lead in the original ore deposits. The decay of uranium into different lead isotopes creates unique isotopic ratios for different lead deposits.
The development of modern analytical instrumentation has focused on the accurate determination of lower and lower concentrations. Techniques have addressed the measurement of major, minor, trace, and ultratrace levels of elements. At each analysis level, these techniques have been concerned primarily with bulk concentrations of the analytes.
My prior U.S. Pat. No. 5,414,259 discloses a method of measuring the elemental species present in a particular sample, not only its bulk elemental concentration. The disclosure of this patent is expressly incorporated herein by reference.
It may be desired to measure a species for a variety of reasons, including characterization and evaluation of systems in environmental science, medicine, biological process monitoring, nutrition, and industry, for example. As the chemistry in these processes is inherently species-specific, the presence of trace elements is measured at the speciated level. For example, Cr(III) is a trace element essential for human health, while Cr(VI) is a poison and carcinogen to both humans and other animals. Each of these forms is a species of chromium, and each has associated with it unique chemical reactions. The difference lies in the elements"" oxidized states. Examples of other species of potential interest are combinations of inorganic ions and covalently bound to organic molecules such as mercury and methylmercury. Others are different chelated species with different ligands and still others are different organic molecules entirely. There are many species described in the chemical literature and a general reference method for distinguishing many of them is highly desirable in order to determine if alterations in their concentrations and relevant abundances have changed during chemical processing and measurement.
Methods of elemental speciation have been known. See Allen, H. E.; Huang, C. P.; Bailey, G. W.; Bowers, A. R. Metal Speciation and Contamination of Soil; Lewis Publisher: Boca Raton, Fla., 1995; Batley, G. E. Trace Element Speciation: Analytical Methods and Problems; CRC Press: Boca Raton, Fla., 1989; Das, A. K.; Chakraborty, R.; Cervera, M. L.; de la Guardia, M. Mikrochim. Acta 1996, 122, 209-246; Kramer, J. R.; Allen, H. E. Metal Speciation: Theory, Analysis and Application; Lewis Publishers: Chelsea, Michigan, 1991; Krull, I. S. Trace Metal Analysis and Speciation; Elsevier: Oxford, 1991; Van Loon, J. C.; Barefoot, R. R. Analyst (London) 1992, 117, 563-570; Vela, N. P.; Olson, L. K.; Caruso, J. A. Anal. Chem. 1993, 65, 585a-597a. Several specific problems that cause errors in speciation analysis are identified in this literature. Currently, only bulk measurements of total element concentrations can be made routinely and accurately. Several potential problems may exist with speciation methods. Many species are reactive, and are transformed or converted to other species during the sampling, storage, and measurement steps. Also, species continue to react during these processes and may be altered many times prior to the numerical measurement. Further, these classical methods do not correct for the species"" possible reaction with separating agents. As a result, although analysis through these methods may be both precise and replicable, the results of such analysis are not fully reliable. For example, the state of California has enacted legislation relating to the analysis of Cr(VI) in water, soils and contaminated wastes, even though there are no fully accurate methods to make these measurements. For regulatory purposes, environmental solutions thus far frequently have been to analyze samples for total chromium and assume all chromium may be in the +6 oxidation state. This may be safe, but it is an unsophisticated solution and wastes a significant amount of money on unnecessary remediation. Other methods with unknown accuracy have also been applied, such as a pair of US EPA RCRA (United States Environmental Protection Agency""s Resource Conservation and Recovery Act) Methods 3060, which is an alkaline extraction for isolating Cr(VI) from soils and solid materials, and 7196, which is an ultraviolet-visible colorimetric method for the quantification of Cr(VI). These methods have biases and are inaccurate in various kinds of sample matrices with no way to evaluate their own accuracy and require another method to validate them. Until now there has been no validation method or way to evaluate bias in these methods. While some methods may be accurate for some matrices and invalid for others there has been up to now no way to tell which and no method of validating a method for specific matrices where it is appropriate.
Traditional methodologies do not accurately analyze species concentrations. For example, results reported in a recent paper noted degradation during the extraction process. To counteract degradation, shorter extraction times were used to extract tin species. Known methods do not provide means for correction for species degradation or correct for or evaluate extraction inefficiency. See Donard, O. F. X.; Lalxc3xa8re, B.; Martin, F.; Lobinski, R. Anal. Chem. 1995, 67, 4250-4254. Methods such as these are used to obtain consensus of species concentration where consistent precision is assumed to be accuracy and where systematic errors are ignored in the certification of standard reference materials. In these cases, errors in standard materials and in the validation of methods used in their certification are reproducible and transferable and tend to become incorporated into both analytical techniques and into standards certified using them. Systematic bias cannot be evaluated due to the fact that no additional degree of freedom exists in these methods to evaluate accuracy of method protocols and standards produced from these protocols.
The traditional analytical methods required a complete extraction prior to analytical detection. To address this balance between extraction efficiency and species degradation requires a technique that is not subject to these quantitative limitations. The decomposition of the matrix to free the species from the sample while preserving the species itself, is a complex task requiring extensive preparation. The separation of one species from the other is also required due to the inability of most detectors to distinguish between species. The conversion of a species, such as Cr(VI) during the analysis process, has been known. For example, EPA Method 3060A (SW-846 EPA Method 3060A: Alkaline Digestion of Hexavalent Chromium, Test Methods for Evaluating Solid Waste, 3rd update; U.S. Environmental Protection Agency: Washington, D.C., 1997) uses alkaline digestion to preserve Cr(VI) and attempt to resist reduction of Cr(VI) to Cr(III) during the extraction process (James, B. R.; Petura, J. C.; Vitale, R. J.; Mussoline, G. R. Environ. Sci. and Tech. 1995, 29, 2377-2381; Vitale, R. J.; Mussoline, G. R.; Peura, J. C.; James, B. R. J. of Environ. Qual. 1994, 23, 1249-1256; Vitale, R. J.; Mussoline, G. R.; Petura, J. C.; James, B. R. Am. Environ. Lab. 1995, 7, 1). The degree of success depends on the matrix and it is not known for which matrices the method is valid. As a result the method is generally employed with known inaccuracies existing. EPA Method 7196A (SW-846 EPA Method 7196A: Chromium, Hexavalent (colorimetric), Test Methods for Evaluating Solid Waste, 3rd ed., U.S. Environmental Protection Agency: Washington, D.C., 1996), a UV-Vis detection method for Cr(VI), has been used extensively to quantify Cr(VI) by detecting the violet-colored complex, Cr(VI)-diphenylcarbazide at pH 2 (Nazario, C. L.; Menden, E. E. J. Am. Leather Chem. Assoc. 1990, 85, 212-224). Several problems arise, however, when this method is applied to samples with complex matrices (Harzdorf, A. C. Int. J. Environ. Anal. Chem. 1987, 29, 249-261; Milacic, R.; Stupar, J.; Kozuh, N.; Korosin, J. Analyst (London) 1992, 117, 125-130). Such coexisting matrix components as Fe2+ and some organic matter, can interfere with the Cr(VI) by reducing it during measurement (SW-846 EPA Method 7196A: Chromium, Hexavalent (colorimetric), Test Methods for Evaluating Solid Waste, 3rd ed., U.S. Environmental Protection Agency: Washington, D.C., 1996). Known methods do not validate these methods for specific matrix and sample types. Similar problems exist for other methods for speciated measurement for other reactive species.
Methods for speciated measurement primarily involve physical separation of species of interest from other forms of the same element and analysis of this subsample (Fong, W.; Wu, J. C. G. Spectrosc. Lett. 1991, 24, 931-941; Beceiro Gonzalez, E.; Bermejo Barrera, P.; Bermejo Barrera, A.; Barciela Garcia, J.; Barciela Alonso, C. J. Anal. At. Spectrom. 1993, 8, 649-653; Peraniemi, S.; Ahlgren, M. Anal. Chim. Acta 1995, 315, 365-370; Beceiro Gonzalez, E.; Barciela Garcia, J.; Bermejo Barrera, P.; Bermejo, B. Fresenius"" J. Anal. Chem. 1992, 344, 301-305). This approach generally assumes that species conversion is negligible in the subsequent processing and analysis steps. However, this is not necessarily the case. Separation of the species of interest from the rest of the matrix can complicate and prolong the analytical procedure. For example, chromatography can separate two different forms of Cr in a mixture before presentation to elemental detectors, such as ICP-MS. As each species can react with its surroundings, and even with the separating agents, detection after a chromatographic separation is only a determination of the species distribution at that latter time and incorporates all altering concentration shifts. Each species may react with other sample components and reagents, or be transformed during the storage and analysis steps (Behne, D. Analyst (London) 1992, 117, 555-557), therefore, there is no reliable way to determine how much chromium was actually present in each speciated form when the sample was originally taken or prior to any manipulation step in the complete method. Such problems lead to biases and inaccuracies that limit the use of these measurements in environmental decision-making and for other purposes, such as use in court, for example.
A review of Analytical Abstracts Database (Royal Society of Chemistry, England) for both chromium and speciation shows that a variety of different approaches have been used for speciated chromium analysis. These are primarily electrochemistry, extraction and chromatography. Electrochemical methods can distinguish between the two different forms of chromium in a simple mixture, based on the different reduction potentials of the respective species, but matrix components can complicate samples, especially from residual organic compounds, and interfere with measurement and may completely observe the measurement in actual environmental samples. (Hassan, S. S. M.; Abbas, M. N.; Moustafa, G. A. E. Talanta 1996, 43, 797-804; Paniagua, A. R.; Vazquez, M. D.; Tascon, M. L.; Sanchez Batanero, P. Electroanalysis (N. Y.) 1993, 5, 155-163; Achterberg, E. P.; Van den Berg, C. M. G. Anal. Chim. Acta 1994, 284, 463-471). For more complex sample matrices, chromatography can physically separate Cr(III) from Cr(VI) before detection (Michalke, B. Fresenius"" J. Anal. Chem. 1996, 354, 557-565; De Smaele, T.; Moens, L.; Dams, R.; Sandra, P. LC GC Int.0 1996, 9, 138-140, 142; Pobozy, E.; Wojasinska, E.; Trojanowicz, M. J. Chromatogr., A 1996, 736, 141-150; Tomlinson, M. J.; Wang, J.; Caruso, J. A. J. Anal. At. Spectrom. 1994, 9, 957-964). In addition, extraction may also be employed. Neither of these two conventional speciation methods, however, can correct for any species transformation occurring before or during measurement.
The analysis of chromium species was chosen as an example for this disclosure. SIDMS is important due to environmental measurements of Cr(VI), as well as other types. Also, only two oxidation states of chromium are predominant in environmental samples, +3 (Cr3+) and +6 (CrO42xe2x88x92 and Cr2O72xe2x88x92), thereby reducing the number of possible reactions. The reaction chemistry of each species has been explored extensively. See, generally, (Harzdorf, A. C. Int. J. Environ. Anal. Chem. 1987, 29, 249-261; Serfass, E. J.; Muraca, R. F. In Chromium: Chemistry of chromium and its compounds; Udy, M. J., Ed.; Reinhold Publishing Corporation: New York, 1956; Vol. I, pp 53-75; Weckhuysen, B. M.; Wachs, I. E.; Schoonheydt, R. A. Chem. Rev. 1996, 96, 3327-3349) and is known to those skilled in the art. In addition, the differentiation between Cr(VI) and Cr(III) is very important because of their vastly different toxicities. Cr(III) is a nutrient known to be important in glucose metabolism, whereas Cr(VI) is highly toxic to humans and animals, causing some types of cancer (Paustenbach, D. J.; Meyer, D. M.; Sheehan, P. J.; Lau, V. Toxicology and Industrial Health 1991, 7, 159-196; Nriagu, J. O.; Nieboer, E. In Advances in Environmental Science and Technology; Nriagu, J. O., Ed.; John Wiley and Sons: New York, 1988; Vol. 20; Burrows, D. Chromium Metabolism and Toxcity; CRC Press, Inc.: Boca Raton, Fla., 1983). This difference in toxicity is important for the type and cost of environmental remediation of contaminated sites. The presence of Cr(III) may require no environmental cleanup, while the presence of Cr(VI) requires remediation. The procedure of the present invention is applicable to samples containing two interconvertable species each of which may also be destroyed or increased. While this disclosure employs only two specific species, SIDMS is not restricted to the measurement of two species, nor of this specific species alone.
In spite of the foregoing prior art knowledge and procedures there remains a very real and substantial need for a method of accurate quantification of reactive species of interest which method compensates for incomplete extraction, separation, isolation, or degradation of species, as well as related inadequacies of the prior art, such as the need for a means to validate methods of unknown accuracy.
The present invention has solved the above-described problems by providing unique methodologies for solution thereto.
The method provides for quantification of one or more species contained in a sample with compensation for species conversion and incomplete separation. In one preferred embodiment, a predetermined stable isotope is converted to a speciated enriched isotope corresponding to the specie to be measured in the same. The sample containing the specie to be measured is spiked and the isotopic spiked species and the species to be measured are brought to equilibrium. All of the species are separated from the sample and isotopic ratios are determined for each specie to be measured. The isotopic ratios are then employed to mathematically deconvolute the species concentration while correcting for species conversion and/or incomplete separation. The method may be practiced on more than one specie to be measured simultaneously. The sample may be an aqueous sample, such as a species containing aqueous solution, for example, or may be a solid sample, such as a soil matrix within which one or more species are located. The method may be practiced in quantifying Cr(III) and Cr(VI) and other species that possess multiple isotopes of the metal or ligand or molecule.
Separation may be accomplished by chromatography, such as time resolution chromatography or other suitable extraction means, such as solvent stabilization or other suitable separation means. Mass spectrometry, such as an ICP mass spectrometer, or other suitable mass spectrometers, may be employed to determine the isotope ratios.
The method of the present invention may also be employed to validate traditional or other methods of speciated analysis. It also may be employed to analyze species and certify standard materials and to prepare speciated spiked standard materials.
It is an object of the present invention to provide a method of measurement of elemental, ionic, molecular or complex species using isotope spiking of species, separation, and isotope dilution fraction measurement to provide a determination of the quantity of the species of interest in the sample.
It is a further object of the invention to provide a method which will effect accurate quantification of the species of interest in spite of incomplete extraction, solubility, separation, isolation or degradation of species.
It is a further object of the present invention to provide a method for validating other methods of unknown accuracy for specific matrix types and for procedural alterations to conserve species.
It is a further object of the present invention to provide such a method that will facilitate correction for incomplete isolation of the species through the use of a tag which joins the isotope spike specie with the specie to be measured.
It is another object of the invention to provide such a method which will permit measurement and correction for phase changes, insoluble transitions and volatile forms of the species while retaining the ability to quantify the species.
It is a further object of the present invention to provide a method to effect quantitative speciated measurement of a specific species in a sample from an unknown fraction of the sample.
It is a further object of the present invention to provide such a method which permits species conversion and corrects for these conversions.
It is a further object of the present invention to provide such a method which not only corrects for species conversions, but improves the precision and detection limits of the measurement.
It is yet another object of the present invention to provide such a method which will permit accurate quantification of the reactive species of interest from a sample despite conversion during the measurement, manipulation, storage or sampling thereof.
It is another object to provide a method to encode standards to permit their use after significant degradation of the species by correcting for the degradation by making SIDMS measurements and correcting the species remaining to their new current ratios and concentrations.
It is another object to permit the manufacturing of speciated standards that have been previously spiked with separated stable isotopes in speciated form that permit the use of these standards subsequently after storage and possible degradation.
It is another object to permit the evaluation of species in standards by evaluation by means of SIDMS to verify the current integrity of these standards as to the current concentration of a single or multiple species.
It is a further object of the present invention to provide such methods which may be employed in validation of other methods of speciated analysis which may be employable with the methods of the present invention.
It is yet another object of the invention to provide methods of analyzing species to permit certification of standard materials including speciated spiked standard materials.
These and other objects of the invention will be more fully understood from the following description of the invention on reference to the illustrations appended hereto.